N-(3,5-dichlorophenyl)succinimide Research Articles

Cytotoxicity of thiazolidinedione-, oxazolidinedione- and pyrrolidinedione-ring containing compounds in HepG2 cells

Liver damage occurred in some patients who took troglitazone (TGZ) for type II diabetes. The 2,4-thiazolidinedione (TZD) ring in TGZ’s structure has been implicated in its hepatotoxicity. To further examine the potential role of a TZD ring in toxicity we used HepG2 cells to evaluate two series of compounds containing different cyclic imides. N-phenyl analogues comprised 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT); 3-(3,5-dichlorophenyl)-2,4-oxazolidinedione (DCPO) and N-(3,5-dichlorophenyl)succinimide (NDPS). Benzylic compounds, which closely resemble TGZ, included 5-(3,5-dichlorophenylmethyl)-2,4-thiazolidinedione (DCPMT); 5-(4-methoxyphenylmethyl)-2,4-thiazolidinedione (MPMT); 5-(4-methoxyphenylmethylene)-2,4-thiazolidinedione (MPMT-I); 5-(4-methoxyphenylmethyl)-2,4-oxazolidinedione (MPMO); 3-(4-methoxyphenylmethyl)succinimide (MPMS) and 3-(4-methoxyphenylmethylene)succinimide (MPMS-I). Cytotoxicity was assessed using the MTS assay after incubating the compounds (0–250μM) with HepG2 cells for 24h. Only certain TZD derivatives (TGZ, DCPT, DCPMT and MPMT-I) markedly decreased cell viability, whereas MPMT had low toxicity. In contrast, analogues without a TZD ring (DCPO, NDPS, MPMO, MPMS and MPMS-I) were not cytotoxic. These findings suggest that a TZD ring may be an important determinant of toxicity, although different structural features, chemical stability, cellular uptake or metabolism, etc., may also be involved. A simple clustering approach, using chemical fingerprints, assigned each compound to one of three classes (each containing one active compound and close homologues), and provided a framework for rationalizing the activity in terms of structure.

Role of leukotrienes in N-(3,5-dichlorophenyl)succinimide (NDPS) and NDPS metabolite nephrotoxicity in male Fischer 344 rats

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) can induce marked nephrotoxicity in rats following a single intraperitoneal (ip) administration of 0.4mmol/kg or greater. Although NDPS induces direct renal proximal tubular toxicity, a role for renal vascular effects may also be present. The purpose of this study was to examine the possible role of vasoconstrictor leukotrienes in NDPS and NDPS metabolite nephrotoxicity. Male Fischer 344 rats (4 rats/group) were administered diethylcarbamazine (DEC; 250 or 500mg/kg, ip), an inhibitor of LTA4 synthesis, 1h before NDPS (0.4mmol/kg, ip), N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS, 0.1, 0.2, or 0.4mmol/kg, ip), or N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA, 0.1mmol/kg, ip) or vehicle. In a separate set of experiments, the LTD4 receptor antagonist LY171883 (100mg/kg, po) was administered 0.5h before and again 6h after NDHS (0.1mmol/kg, ip) or 2-NDHSA (0.1mmol/kg, ip) or vehicle. Renal function was monitored for 48h post-NDPS or NDPS metabolite. DEC markedly reduced the nephrotoxicity induced by NDPS and its metabolites, while LY171883 treatments provided only partial attenuation of NDHS and 2-NDHSA nephrotoxicity. These results suggest that leukotrienes contribute to the mechanisms of NDPS nephrotoxicity.

Nephrotoxicity induced by N‐(3,5‐dichlorophenyl)‐3‐hydroxysuccinamic acid in male and female Fischer 344 rats

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) is a more potent nephrotoxicant in female rats than in males. Similarly, nephrotoxicant NDPS metabolites studied to date in male and female rats have also demonstrated gender differences, being twice as potent as nephrotoxicants in females as in males. The purpose of this study was to examine the nephrotoxic potential of N-(3,5-dichlorophenyl)-3-hydroxysuccinimide (3-NDHSA) in male and female Fisher 344 rats to determine if gender differences in nephrotoxic potential also exist for this metabolite. Rats (four per group) were administered a single intraperitoneal (i.p.) injection of 3-NDHSA (0.1, 0.2 or 0.4 mmol kg(-1)) or vehicle, and renal function was monitored at 24 and 48 h. 3-NDHSA 0.1 mmol kg(-1) did not induce nephrotoxicity in male or female rats. In male rats, 3-NDHSA 0.2 mmol kg(-1) induced mild nephrotoxicity seen as diuresis and transient, mild proteinuria. However, 3-NDHSA 0.4 mmol kg(-1) induced marked nephrotoxicity. In female rats, 3-NDHSA 0.2 mmol kg(-1) induced mild nephrotoxicity, as evidenced by transient diuresis and proteinuria. As in males, 3-NDHSA 0.4 mmol kg(-1) induced marked nephrotoxicity. These results indicate that, unlike NDPS and other nephrotoxic NDPS metabolites, 3-NDHSA does not exhibit gender differences in nephrotoxic potential. In addition, in comparison with NDPS and other nephrotoxic NDPS metabolites, 3-NDHSA is a less potent nephrotoxicant that NDHS or 2-NDHSA and similar to NDPS in nephrotoxic potential in male rats.

Nephrotoxicity induced by the R- and S-enantiomers of N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and their sulfate conjugates in male Fischer 344 rats

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity characterized as polyuric renal failure and mediated via metabolites arising from oxidation of the succinimide ring. Recent findings have suggested that the stereochemical nature of NDPS metabolites may be an important factor in NDPS metabolite-induced nephrotoxicity. The purpose of the present study was to determine the role of stereochemistry in the in vivo nephrotoxicity induced by R-(+)- and S-(−)- N-(3,5-dichlorophenyl)-2-hydroxysuccinimide ( R- and S-NDHS) and the in vitro nephrotoxicity induced by their enantiomeric sulfate conjugates, R-(−)- and S-(+)- N-(3,5-dichlorophenyl)-2-hydroxysuccinimide- O-sulfate ( R- and S-NSC). Male Fischer 344 rats (four rats/group) were administered intraperitoneally (i.p.) an enantiomer of NDHS (0.05, 0.1 or 0.2 mmol/kg) or vehicle, and renal function monitored for 48 h. R-NDHS (0.1 or 0.2 mmol/kg) had little effect on renal function. In contrast, S-NDHS (0.1 mmol/kg) induced marked nephrotoxicity. The nephrotoxic potential of R- and S-NSC (0.5, 0.75 or 1.0 mM) was determined using freshly isolated rat renal cortical cells (IRCC, 3–4 × 10 6 cells/ml). Cytotoxicity was determined by measuring the release of lactate dehydrogenase (LDH) at the end of a 1 h incubation period. The LDH release observed in these studies was similar between R- and S-NSC. These results indicate that stereochemistry is an important factor for NDPS metabolite nephrotoxicity and that the role of stereochemistry, at least for NSC, occurs at extra-renal sites.

Effect of gender on phase I and phase II metabolism of N‐(3,5‐dichlorophenyl)succinimide (NDPS)

NDPS is a model compound for examining chemically-induced kidney damage. The parent compound and its direct oxidative metabolite, N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS), induce nephrotoxicity in rats. Toxicity of NDPS is thought to require hepatic formation of NDHS followed by conjugation to reactive phase II metabolites. Further studies have shown that female rats are more susceptible to toxicity than males. In this study, the relationship between turnover and gender was examined. Metabolism was studied by incubating rat liver microsomes with the appropriate substrate and cofactor (oxidation: NDPS, NADPH; glucuronidation: NDHS, UDPGA) for time periods between 15 and 120 min. Samples were analyzed by HPLC with UV detection or HPLC-ESI-MS, and relative metabolite formation was determined. Results indicate that females are capable of producing the same oxidative metabolites and glucuronide conjugates that were previously found in males. A quantitative difference in the formation of oxidative metabolites was not observed. However, NDHS glucuronidation was significantly higher in male microsomes at incubation periods of 60 min or less. Whether this is relevant to the differential susceptibility of the genders to NDPS nephrotoxicity remains to be determined. Other phase II pathways, such as sulfation, may also vary between female and male rats. Supported by PHS grant ES012499.

Transamination in the Metabolism of the Nephrotoxicant N-(3,5-Dichlorophenyl)succinimide in Rats

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) is nephrotoxic in rats. Due to the involvement of NDPS metabolism in its mechanism of toxicity, the detailed biotransformation of 14C-NDPS in rats was previously evaluated using high-performance liquid chromatography-electrospray ionization-mass spectrometry. In the present report, we describe the identification of two novel amino metabolites of NDPS, which were present in significant amounts in rat kidney tissues. Using liquid chromatography-tandem mass spectrometry and synthetic standards, the two metabolites were identified as N-(3,5-dichlorophenyl)-2-aminosuccinamic acid (2-NDASA) and its N-acetylated derivative (N-acetyl-2-NDASA). The mechanism of formation of 2-NDASA was studied in vitro. Incubations were carried out in rat liver and kidney cytosols using the major oxidative metabolite of NDPS, N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid, as the substrate. Formation of 2-NDASA in vitro was confirmed using mass spectrometry. Inhibitors of alcohol dehydrogenase (4-methylpyrazole) and aldehyde dehydrogenase (disulfiram) reduced 2-NDASA formation by 40 to 50%. Menadione (an inhibitor of aldehyde oxidase) and quercetin (an inhibitor of carbonyl reductase) did not show any effects. (Aminooxy)acetic acid, an inhibitor of pyridoxal 5'-phosphate-containing enzymes such as aminotransferases, almost completely abolished the formation of 2-NDASA. Using liquid chromatography-mass spectrometry, the transamination mechanism was further supported by the incorporation of a 15N-amino group in 2-NDASA when 15N-glutamic acid was included in the incubation mixture. Results from these studies show that transamination is a metabolic pathway in the clearance of NDPS in rats, and that cytosolic dehydrogenases and aminotransferases may be involved in this process.

Metabolism of the Nephrotoxicant N-(3,5-Dichlorophenyl)succinimide in Rats: Evidence for Bioactivation through Alcohol-O-glucuronidation and O-Sulfation

The agricultural fungicide, N-(3,5-dichlorophenyl)succinimide (NDPS), was shown to be nephrotoxic in rats. Previous studies have indicated that the metabolism of NDPS contributes to its mechanism of toxicity and both phase I and phase II pathways may be involved. In the current report, we investigated the detailed biotransformation of [(14)C]NDPS in rats using HPLC-ESI-MS. The chemical reactivity of the phase II NDPS metabolites was also evaluated. In vivo studies were conducted by administering [(14)C]NDPS to male Fischer 344 rats. Urine, tissue (liver and kidney), and plasma samples were analyzed. The mechanism of formation and chemical reactivity of the glucuronide and sulfate metabolites of NDPS were investigated in vitro using liver subcellular preparations. Major in vivo metabolites of NDPS were identified as the oxidative [N-(3,5-dichlorophenyl)-2- and 3-hydroxysuccinamic acid, 2-/3-NDHSA] and hydrolytic products [N-(3,5-dichlorophenyl)succinamic acid]. N-Acetylcysteine and cysteine (with intramolecular aminolysis) conjugates were also detected in rat urine and fecal extracts, respectively, suggesting the formation of reactive intermediate(s) in the metabolism of NDPS. Small amounts of the alcohol-O-glucuronide and O-sulfate of 2-/3-NDHSA were detected in rat urine, plasma, and tissue homogenates. The formation of these phase II metabolites was found to be mediated through the initial conjugation of N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) followed by hydrolysis. As compared to NDHS, NDHS-O-sulfate is approximately 500-fold more reactive toward GSH conjugation. In rat liver S9, fortifying phase II cofactors (UDPGA or PAPS) in incubation mixtures with NDHS also significantly increased the amount of GSH adducts produced. Results of this research demonstrate that phase II metabolites of NDPS were produced in rats. The formation of the alkyl alcohol-O-glucuronide and O-sulfate conjugates represents bioactivation pathways in the metabolism of NDPS that could potentially contribute to its mechanism of nephrotoxicity.

Nephrotoxic and hepatotoxic potential of imidazolidinedione-, oxazolidinedione- and thiazolidinedione-containing analogues of N-(3,5-dichlorophenyl)succinimide (NDPS) in Fischer 344 rats

Nephrotoxicity of the agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) in rats is believed to involve metabolism on the succinimide ring. To further investigate this hypothesis, we synthesized and tested the following NDPS analogues, which contain other cyclic imide rings and may therefore be metabolized differently than NDPS: 3-(3,5-dichlorophenyl)-2,4-oxazolidinedione (DCPO), 3-(3,5-dichlorophenyl)-2,4-imidazolidinedione (DCPI), 3-(3,5-dichlorophenyl)-1-methyl-2,4-imidazolidinedione (DCPM) and 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT). Male Fischer 344 rats were administered DCPO, DCPI, DCPM, DCPT (0.6 or 1.0 mmol/kg, i.p. in corn oil), NDPS (0.6 mmol/kg, i.p. in corn oil) or corn oil (4 ml/kg). As evidenced by diuresis, proteinuria, elevated blood urea nitrogen levels, increased kidney weights and proximal tubular damage, NDPS produced severe nephrotoxicity in the rats. In contrast, DCPO, DCPI, DCPM and DCPT were mild nephrotoxicants. None of the compounds elevated serum alanine transferase activity or liver weights in the rats, however DCPT produced centrilobular necrosis. These experiments confirm that NDPS-induced nephrotoxicity is critically dependent on the presence of the succinimide ring. Furthermore, replacement of the succinimide ring with a thiazolidinedione ring produced a more pronounced effect on the liver than on the kidney. Liver damage has been reported in type II diabetic patients taking troglitazone, rosiglitazone and pioglitazone. Since these compounds also contain a thiazolidinedione ring, DCPT may be useful for investigating the role of this structural feature in hepatotoxicity.

EFFECT OF THREE N -ACETYLAMINO ACIDS ON N -(3,5-DICHLOROPHENYL)SUCCINIMIDE (NDPS) AND NDPS METABOLITE NEPHROTOXICITY IN FISCHER 344 RATS

The agricultural fungicide N -(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity in mammals characterized as polyuric renal failure and proximal tubular necrosis. Recent studies have suggested that NDPS-induced nephrotoxicity may be mediated by metabolites arising from the nephrotoxic NDPS metabolites N -(3,5-dichlorophenyl)2-hydroxysuccinimide (NDHS) and/or N -(3,5-dichlorophenyl)-2-succinamic acid (2-NDHSA). The purpose of this study was to examine the effects of N -acetylcysteine (NAC), a nucleophilic agent, and two nonnucleophilic N -acetylamino acids, N -acetylserine (NAS) and N -acetylalanine (NAA), on NDPS and NDPS metabolite-induced nephrotoxicity. Male Fischer 344 rats (4-8/group) were administered intraperitoneally (ip) an N -acetylamino acid (1 mmol/kg) 2 h before an ip injection of NDPS (0.4 mmol/kg), NDHS (0.1 mmol/kg), 2-NDHSA (0.1 mmol/kg), or vehicle. Renal function was then monitored at 24 and 48 h. NAC pretreatment markedly attenuated NDPS-, NDHS-, and 2-NDHSA-mediated nephrotoxicity. The nonnucleophilic N -acetylamino acids (NAS, NAA) only partly reduced NDPS and NDHS nephrotoxicity, and they had little effect on 2-NDHSA nephrotoxicity. These results suggest that reactive NDPS metabolites may be formed from NDHS and 2-NDHSA and that nucleophilic substrates (e.g., NAC) may offer protection from NDPS-induced nephrotoxicity. However, mechanisms other than chemical neutralization of reactive NDPS metabolites may also be contributing to the attenuation of NDPS nephrotoxicity, since nonnucleophilic N -acetylamino acids (e.g., NAA) also provided some protection against NDPS and NDHS nephrotoxicity.

Role of stereochemistry in N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA) nephrotoxicity

The nephrotoxicity induced by the agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) is mediated through oxidative metabolites of NDPS. Oxidation of the succinimide ring in NDPS yields the nephrotoxic metabolites N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and its hydrolysis product N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA). The oxidation of NDPS on the succinimide ring also introduces an asymmetric carbon atom into these NDPS metabolites, so that R- and S- enantiomers of NDHS and 2-NDHSA are possible. The purpose of this study was to begin to explore the importance of the stereochemical orientation at the asymmetric carbon atom for the nephrotoxicity induced by NDPS metabolites. Male Fischer 344 rats were administered a single intraperitoneal (ip) injection of R-(+)- or S-(−)-2-NDHSA (0.05, 0.1 or 2.0 mmol/kg) or vehicle, and renal function was monitored for 48 h. R-2-NDHSA (0.1 mmol/kg) administration had little effect on renal function. R-2-NDHSA (0.2 mmol/kg) treatment induced mild diuresis on day 1, increased proteinuria, and a small increase in blood urea nitrogen (BUN) concentration, but no change in kidney weight or glucosuria. S-2-NDHSA (0.1 mmol/kg) induced marked nephrotoxicity as evidenced by diuresis on both post-treatment days, increased proteinuria, glucosuria, and increased kidney weight and BUN concentration. No evidence of hepatotoxicity was obtained in any treated group. Thus, the S-isomer of 2-NDHSA is a more potent nephrotoxicant than the R-isomer, and stereochemistry may play a role in NDPS metabolite-induced nephrotoxicity.

GENDER DIFFERENCES IN THE POTENTIATION OF N-(3,5-DICHLOROPHENYL)SUCCINIMIDE METABOLITE NEPHROTOXICITY BY PHENOBARBITAL

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces acute nephrotoxicity characterized as polyuric renal failure with proximal tubular necrosis. Phenobarbital pretreatment potentiates NDPS and N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS, a nephrotoxic metabolite of NDPS) nephrotoxicity in male rats. The purpose of this study was to determine the ability of phenobarbital pretreatment to potentiate (1) NDHS nephrotoxicity in female rats and (2) N -(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA, a nephrotoxic metabolite of NDHS) nephrotoxicity in male and female rats. Age-matched male and female Fischer 344 rats (4/group) were pretreated intraperitoneally (ip) with phenobarbital (75 mg/d, 3 d). At 24 h after the last injection of phenobarbital, an ip injection of NDHS (0.025 mmol/kg), 2-NDHSA (0.025 mmol/kg, females; 0.05 mmol/kg, males), or vehicle was given and renal function was monitored at 24 and 48 h post NDPS metabolite or vehicle. Additional groups received the NDPS metabolite or vehicle only and were also monitored for 48 h. In a separate experiment, male Fischer 344 rats were pretreated with piperonyl butoxide (PIBX, 1360 mg/kg) or the PIBX vehicle. 2-NDHSA (0.1 mmol/kg) or vehicle was administered (ip) 30 min after PIBX, and renal function was monitored for 24 h. Phenobarbital markedly potentiated 2-NDHSA nephrotoxicity in male rats as evidenced by increased kidney weight, increased blood urea nitrogen (BUN) concentration, and decreased tetraethylammonium (TEA) accumulation by renal cortical slices. PIBX had no effect on 2-NDHSA nephrotoxic ity. Phenobarbital pretreatment did not markedly enhance the nephrotoxic potential of NDHS or 2-NDHSA in female rats. These results indicate that phenobarbital exhibits differential potentiation of NDPS metabolite nephrotoxicity in male and female rats and that the potentiation of 2-NDHSA nephrotoxicity observed in males is not due to cytochrome P-450-mediated oxidative biotransformation.

In vitro nephrotoxicity induced by N-(3,5-dichlorophenyl)succinimide (NDPS) metabolites in isolated renal cortical cells from male and female Fischer 344 rats: evidence for a nephrotoxic sulfate conjugate metabolite

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity in vivo that is characterized as acute polyuric renal failure and proximal tubular necrosis. However, earlier in vitro studies have failed to reproduce the in vivo nephrotoxicity seen with NDPS or its nephrotoxic metabolites N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA). The purpose of this study was to examine the nephrotoxic potential of NDPS, its known non-conjugated metabolites, the O-sulfate conjugate of NDHS (NSC), and the putative metabolite N-(3,5-dichlorophenyl)maleimide (NDPM) and its hydrolysis product N-(3,5-dichlorophenyl)maleamic acid (NDPMA) using freshly isolated renal cortical cells (IRCC). IRCC were obtained from untreated male or female Fischer 344 rats following collagenase perfusion of the kidneys. Cells (∼4 million per ml) ( N=4) were incubated with up to 1.0 mM NDPS or an NDPS metabolite or vehicle for up to 120 min. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release into the medium. Only NSC (>0.5 mM) and NDPM (≥0.5 mM) exposure increased LDH release from IRCC. NSC 1.0 mM or NDPM 0.5 mM increased LDH release from IRCC within 15–30 min of exposure. NDPS or the remaining NDPS metabolites did not increase LDH release at bath concentrations of 1.0 mM for exposures of 120 min. IRCC from male and female rats responded similarly to the toxic effects of NDPS and its metabolites. These results demonstrate that sulfate conjugates of NDPS metabolites can be fast acting nephrotoxicants and could contribute to NDPS nephrotoxicity in vivo. These results also suggest that the kidney probably accumulates toxic sulfate conjugates of NDPS metabolites rather than forming the conjugates. In addition, mechanisms responsible for gender differences in nephrotoxicity seen with NDPS and NDPS metabolites in vivo either occur prior to renal accumulation of sulfate conjugates and/or represent biochemical/physiological differences between the genders.

Determination of the site of glucuronidation in anN-(3,5-dichlorophenyl)succinimide metabolite by electrospray ionization tandem mass spectrometry following derivatization to picolinyl esters

Derivatization using 3-pyridylcarbinol coupled with liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) was used to characterize a novel Phase II metabolite of the nephrotoxic agricultural fungicide, N-(3,5-dichlorophenyl)succinimide (NDPS). A glucuronide conjugate of N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA) was identified in the urine from a rat dosed with [14C]NDPS. However, 2-NDHSA contains an aliphatic hydroxyl group and a carboxylic acid group, both of which are potential sites for glucuronidation. Mass spectrometry alone was unable to distinguish between these possibilities. Since the position of glucuronidation may be important in the mechanism of NDPS-induced nephrotoxicity, chemical derivatization in conjunction with mass spectrometry was used to characterize the glucuronide. The 2-NDHSA glucuronide conjugate was isolated from rat urine, derivatized with 3-pyridylcarbinol, and the derivatized metabolite was then analyzed by LC/MS/MS. Two known NDPS metabolites, 2-NDHSA and N-(3,5-dichlorophenyl)succinamic acid (NDPSA), were also isolated from rat urine and derivatized similarly. 3-Pyridinylcarbinol reacted rapidly with the carboxylic acid groups and formation of the picolinyl esters increased the ionization potential under positive ion conditions. The urinary glucuronide of 2-NDHSA was identified as an alcohol-linked glucuronide by examination of the molecular ions and the collision-induced dissociation (CID) product ion spectra of the derivatized products. When used in combination with mass spectrometry, derivatization of carboxylic acids with 3-pyridylcarbinol provided useful mass fragmentations and is a rapid way to obtain structural information about the position of glucuronidation of NDPS metabolites.

Sodium sulfate potentiates N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA) nephrotoxicity in the Fischer 344 rat

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity as its major toxicity in rats. Previous studies have shown that NDPS induces nephrotoxicity following oxidation of the succinimide ring to form N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and the hydrolysis product of NDHS, N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA). Our recent work found that sodium sulfate potentiated NDPS nephrotoxicity, suggesting that sulfate conjugation of NDPS metabolites might be a bioactivation step mediating NDPS nephrotoxicity. The purpose of this study was to determine if sodium sulfate also potentiated the nephrotoxicity of the two nephrotoxic metabolites of NDPS and further to see if sodium sulfate potentiated NDHS and 2-NDHSA nephrotoxicity to the same degree. Male Fischer 344 rats (4–16 rats/group) received an intraperitoneal (ip) injection of sodium sulfate (10 mg/kg) 20 min before a non-nephrotoxic dose (0.05 mmol/kg, ip) of NDHS or 2-NDHSA, or vehicle (12.5% dimethyl sulfoxide in sesame oil). Renal function was then monitored over 48 h. Sodium sulfate pretreatment potentiated the renal effects of a non-nephrotoxic dose of NDHS and 2-NDHSA to induce nephrotoxicity. Nephrotoxicity was characterized by diuresis, increased proteinuria, elevated blood urea nitrogen (BUN) concentration, increased kidney weight and proximal tubular necrosis. Differences in the potentiation of NDHS and 2-NDHSA nephrotoxicity by sodium sulfate were also observed as NDHS nephrotoxicity was potentiated to a lesser degree than 2-NDHSA-induced nephrotoxicity. These results support the likelihood that one or more sulfate conjugate(s) of NDPS metabolites contribute to NDPS nephrotoxicity.

EFFECT OF SULFATION SUBSTRATES/INHIBITORS ON N-(3,5-DICHLOROPHENYL)SUCCINIMIDE NEPHROTOXOCITY IN FISCHER 344 RATS

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) is an acute nephrotoxicant in rats. Our previous studies suggested that sulfate conjugation of NDPS metabolites might be a bioactivation step mediating NDPS nephrotoxicity. In this study, effects of substrates and/or inhibitors of sulfation on NDPS nephrotoxicity were examined to explore further the role of sulfation in NDPS nephrotoxicity. Male Fischer rats (4-8/group) were administered one of the following intraperitoneal (ip) pretreatment (dose, pretreatment time) prior to NDPS (0.6 mmol/kg) or NDPS vehicle (sesame oil, 2.5 ml/kg): (1) no pretreatment, (2) dehydroepiandrosterone (DHEA) (0.5 mmol/kg, 1 h), or (3) 2,6-dichloro4-nitrophenol (DCNP) (0.04 mmol/kg, 1 h). Following NDPS or NDPS vehicle administration, renal function was monitored at 24 and 48 h. Pretreatment with DHEA, a typical substrate for and an inhibitor of hydroxysteroid (alcohol) sulfotransferase, resulted in marked protection against NDPS nephrotoxicity. A selective inhibitor of phenol sulfotransferase, DCNP, afforded little attenuation in NDPS nephrotoxicity. These results suggest that alcohol sulfate conjugates of NDPS metabolites, rather than phenolic sulfate conjugates, may be a penultimate or ultimate nephrotoxicant species mediating NDPS nephrotoxicity. The marked, but not complete, protection by DHEA also suggests that there are other metabolites or mechanisms responsible for NDPS nephrotoxicity.

Nephrotoxic potential of N-(3,5-dichloro-4-fluorophenyl)succinimide in Fischer 344 rats: comparison with N-(3,4,5-trichlorophenyl)succinimide

Numerous structure–nephrotoxicity relationship studies from our laboratory have demonstrated that N-(3,5-dichlorophenyl)succinimide (NDPS) is one of the most potent nephrotoxicants among the N-arylsuccinimides. The purpose of this study was to extend our previous structure–nephrotoxicity relationship studies by examining the effect of addition of a fluoro verses a chloro group at the 4-phenyl position in NDPS. Male Fischer 344 rats (four rats/group) received a single intraperitoneal (i.p.) injection of N-(3,5-dichloro-4-fluorophenyl)succinimide (NDCFPS) or N-(3,4,5-trichlorophenyl)succinimide (NTCPS)(0.4 or 0.8 mmol/kg) or vehicle, and renal function monitored at 24 and 48 h. NDCFPS did not induce significant nephrotoxicity at either dose tested. In contrast, NTCPS (0.4 or 0.8 mmol/kg) induced marked nephrotoxicity characterized by diuresis, increased proteinuria, glucosuria, elevated kidney weight and increased blood urea nitrogen (BUN) concentration. NTCPS also induced marked proximal tubular necrosis at both doses tested. Neither NDCFPS nor NTCPS induced hepatotoxicity at either dose tested. The results of these experiments indicate that addition of a fluoro group at the 4-position on the phenyl ring of NDPS produces a nonnephrotoxicant NDPS derivative (NDCFPS), while addition of a chloro group at this site produces an NDPS derivative with similar nephrotoxic potential to NDPS. The mechanism for this differential effect between 4-halophenyl substitution is unclear, but may result from increased hydrolysis of the succinimide ring and/or increased clearance of N-arylsuccinimide metabolites when a fluoro group is added to the 4-position of the phenyl ring.

Effect of glucuronidation substrates/inhibitors on N-(3,5-dichlorophenyl)succinimidenephrotoxicity in Fischer 344 rats

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) is an acute nephrotoxicant in rats. Our previous studies have strongly suggested that glucuronide conjugation of NDPS metabolites might be a bioactivation step mediating NDPS nephrotoxicity. In this study, effects of substrates and/or inhibitors of primarily glucuronidation on NDPS nephrotoxicity were examined to explore further the role of glucuronidation in NDPS nephrotoxicity. Male Fischer 344 rats (4–6/group) were administered one of the following intraperitoneal (i.p.) pretreatments (dose, pretreatment time) prior to NDPS (0.4 mmol/kg) or NDPS vehicle (sesame oil, 2.5 ml/kg): (1) no pretreatment; (2) borneol (900 mg/kg, 30 min); (3) eugenol (500 mg/kg per day, 3 days); (4) clofibric acid (400 mg/kg, 15 min before (1/2 dose) and 3 h after (1/2 dose)), or (5) valproic acid, sodium salt (1.0 mmol/kg, 15 min). Following NDPS or NDPS vehicle administration, renal function was monitored at 24 and 48 h. Pretreatment with borneol or eugenol, substrates for ether glucuronidation and sulfation (mainly glucuronidation), afforded complete protection against NDPS nephrotoxicity. Substrates for acyl glucuronidation, clofibric acid or valproic acid, mildly reduced or had little effect on NDPS nephrotoxicity, respectively. These results suggest that ether glucuronide conjugates of NDPS metabolites, rather than acyl glucuronide conjugates, may be the primary ultimate nephrotoxicant species mediating NDPS nephrotoxicity.

GENDER DIFFERENCES IN ACUTE N -(3,5- DICHLOROPHENYL)-2-HYDROXYSUCCINIMIDE (NDHS) AND N -(3,5-DICHLOROPHENYL)-2-HYDROXYSUCCINAMIC ACID (2-NDHSA) NEPHROTOXICITY IN FISCHER 344 RATS

N -(3,5-Dichlorophenyl)succinimide (NDPS) is an agricultural fungicide that induces nephrotoxicity as its major toxicity. NDPS is also a more potent nephrotoxicant in female than in male rats. The purpose of this study was to examine the nephrotoxic potential of the two NDPS metabolites N -(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N -(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA) in agematched male and female Fischer 344 rats to determine if gender differences exist for the nephrotoxicity induced by the two NDPS metabolites. Rats (4 per group) were administered a single intraperitoneal (ip) injection of NDHS or 2-NDHSA (0.025 or 0.05 mmol/kg) or vehicle, and renal function was monitored for 48 h. Neither compound induced significant nephrotoxicity in male rats at the doses tested. However, in female rats both metabolites induced marked nephrotoxicity at the 0.05 mmol/kg dose level, and treatment with 0.025 mmol/kg 2-NDHSA induced some changes in renal function (transient diuresis, transient proteinuria, decreased organic ion accumulation). Little effect on renal function was induced in females by treatment with 0.025 mmol/kg NDHS. At toxic levels in female rats, the renal lesions were located primarily in the S2 and S3 segments of the proximal tubule. These results indicate that, like the parent compound, gender differences exist in the nephrotoxic potential of NDHS and 2-NDHSA. The results also suggest that in females, as in males, NDPS nephrotoxicity is mediated via NDHS and/or 2-NDHSA. However, it is not clear if the ultimate nephrotoxicant species following NDPS exposure is different in males and females or if the same ultimate nephrotoxicant species is produced in both species but handled differently by male and female kidneys. Thus, further studies are needed to determine the exact nature of the ultimate nephrotoxicant species and the mechanisms of the observed gender differences.

Effects of sodium sulfate on acute N-(3,5-dichlorophenyl)succinimide (NDPS) nephrotoxicity in the Fischer 344 rat

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces acute polyuric renal failure in rats. Results of previous studies have suggested that NDPS may induce nephrotoxicity via conjugates of NDPS metabolites. Thus, the purpose of this study was to examine if administered sodium sulfate could alter NDPS nephrotoxicity. Male Fischer 344 rats (four rats per group) were administered a single intraperitoneal (i.p.) injection of sodium sulfate (0.035, 0.07, 0.35 or 3.5 mmol/kg) or sodium chloride (7.0 mmol/kg) 20 min before NDPS (0.2, 0.4 or 0.8 mmol/kg) or NDPS vehicle (sesame oil, 2.5 ml/kg) and renal function monitored at 24 and 48 h. High dose sodium sulfate (3.5 mmol/kg) markedly attenuated NDPS nephrotoxicity, while sodium chloride had no effect on NDPS-induced renal effects. NDPS nephrotoxicity was also attenuated by a pretreatment dose of 0.35 mmol/kg sodium sulfate, while 0.07 mmol/kg sodium sulfate pretreatment potentiated NDPS 0.2 mmol/kg to produce nephrotoxicity without markedly attenuating NDPS 0.4 mmol/kg to induce renal effects. A dose of 0.035 mmol/kg sodium sulfate did not potentiate NDPS 0.2 mmol/kg to induce nephrotoxicity. These results suggest that sulfate conjugates of NDPS metabolites might contribute to NDPS nephrotoxicity.

Cytochrome P450-mediated metabolism and nephrotoxicity of N-(3,5-dichlorophenyl)succinimide in Fischer 344 rats.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) is nephrotoxic in rats. Previous studies have suggested that oxidative hepatic biotransformation is required for the induction of kidney damage. The experiments described in this paper were designed to further investigate the relationship between NDPS metabolism and nephrotoxicity using various modulators of cytochrome P450 activity. Male Fischer 344 rats were pretreated with the P450 inducers Aroclor 1254 (ARO), isoniazid (INH), 3-methylcholanthrene (3-MC), and phenobarbital (PB), or the P450 inhibitor 1-aminobenzotriazole (ABT). Control animals received vehicle only. NDPS metabolism was investigated using hepatocytes isolated from the various treatment groups. Separate experiments were also conducted to evaluate the effects of these pretreatments on NDPS-induced nephrotoxicity in rats. PB and ARO enhanced formation of the known nephrotoxic NDPS metabolites, N-(3,5-dichlorophenyl)-2-hydroxysuccinimide, N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid, and N-(3,5-dichlorophenyl)-3-hydroxysuccinamic acid, by the hepatocytes. In contrast, ABT inhibited formation of the nephrotoxic metabolites, whereas INH and 3-MC did not alter NDPS biotransformation. NDPS-induced renal damage was potentiated by pretreating the rats with PB or ARO and was attenuated by ABT. Compared with control animals, toxicity was unaffected by INH or 3-MC pretreatments. Thus, there was a correlation between pretreatments that induce P450-mediated NDPS metabolism and the effects that these compounds have on NDPS-induced nephrotoxicity. The data indicate that specific P450 isozymes metabolize NDPS to its hydroxylated products and suggest that these metabolites mediate the nephrotoxicity induced by NDPS.